Combinations of cellular immunotherapies

Abstract

A method for treating a tumor, characterized by administering to an individual having a tumor immune effector cells expressing a receptor recognizing a tumor antigen and gemcitabine. A kit for treating a tumor, characterized by comprising: 1) immune effector cells expressing a receptor recognizing a tumor antigen; 2) gemcitabine; 3) a container for containing the above 1) and 2); and 4) a written notice for treating a tumor using the kit.

Description

Technical Field

[0001] This invention belongs to the field of cell immunotherapy, specifically involving the combined use of immune effector cells and gemcitabine for anti-tumor therapy. Background Technology

[0002] In recent years, cellular immunotherapy, such as CAR-T cell therapy, has shown remarkable therapeutic effects in the treatment of hematological malignancies, with over 200 clinical trials currently investigating its use in this field (Clinical development of CAR T cells - challenges and opportunities in translating innovative treatment concepts, Jessica Hartmann et al., EMBO Molecule Medicine, Published online, August 1, 2017). However, its effectiveness in treating solid tumors has not been as significant as in treating hematological malignancies.

[0003] This is because, for hematologic malignancies, CAR-T cells, administered intravenously, can more easily reach and kill tumor cells, while CAR-T cells face greater difficulty in homing to the tumor tissue of solid tumors. Furthermore, solid tumors typically possess a complex and dynamic tumor microenvironment, enabling interactions between tumor cells, benign cells, stromal cells, and vascular cells. Additionally, the tumor microenvironment contains networks of interacting cytokines and growth factors. Therefore, CAR-T therapy is generally less effective when used to treat solid tumors. Summary of the Invention

[0004] The purpose of this invention is to provide a tumor treatment method to improve the efficacy of immune cell therapy, especially CAR-T cell therapy, in solid tumors.

[0005] A first aspect of the present invention provides a method for treating tumors, comprising administering immune effector cells and gemcitabine to an individual suffering from a tumor, said immune effector cells expressing receptors that recognize tumor antigens.

[0006] A second aspect of the invention also provides a method for reducing the growth, survival, or viability of cancer cells, comprising administering immune effector cells and gemcitabine to an individual suffering from a tumor, said immune effector cells expressing receptors that recognize tumor antigens.

[0007] In a preferred embodiment, the immune effector cells and gemcitabine are administered in any order; gemcitabine may be administered first, followed by the immune effector cells; they may be administered simultaneously; or the immune effector cells may be administered first, followed by gemcitabine. Specifically, the method of administering immune effector cells and gemcitabine to an individual with a tumor is selected from any of the following: (1) administering gemcitabine first, followed by the immune effector cells; (2) administering both immune effector cells and gemcitabine simultaneously; and (3) administering immune effector cells first, followed by gemcitabine.

[0008] In another preferred embodiment, the receptor is selected from: chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), or a combination thereof.

[0009] In another preferred embodiment, the tumor antigen is a solid tumor antigen.

[0010] In another preferred embodiment, the tumor antigen is selected from any of the epidermal growth factor receptor family and its mutants (i.e., EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin18.2, Claudin18.1, Claudin 6, phosphatidylinositol proteoglycan-3 (GPC3), and vascular endothelial growth factor receptor.

[0011] In another preferred embodiment, the tumor antigen is selected from: thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD 123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin-13 receptor subunit α (IL-13Rα); Interleukin-11 receptor α (IL-11Rα); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryonic antigen-4 (SSEA-4); Cell surface-associated Mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); LMP2; Hepatic glycoside A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; High molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); Folate receptor; Tumor vascular endothelial marker 1 (TEM1 / CD248); Tumor vascular endothelial marker 7-related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tendinin; carcinoembryonic variant in tumor necrosis zone; G protein-coupled receptor class C5-member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);GloboH (the hexose moiety of glycoceramide); Breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenaline receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms' blastoma protein (WT1); ETS translocation variant gene 6 (ETV6-AML); Sperminin 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 ( MAD-CT-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (Rho C); Cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); Squamous cell carcinoma antigen 3 recognized by T cells (SART3); Pairing box protein Pax-5 (PAX5); Proacrosin-binding protein sp32 (OYTES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); Synovial sarcoma, X-breakpoint 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LA) IR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLL1).

[0012] In another preferred embodiment, the chimeric antigen receptor has:

[0013] (i) Antibodies or fragments thereof that recognize tumor antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD28, and CD3ζ; or

[0014] (ii) Antibodies or fragments thereof that recognize tumor antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD137, and CD3ζ; or

[0015] (iii) Antibodies or fragments thereof that recognize tumor antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD28, co-stimulatory signaling domains of CD137, and CD3ζ.

[0016] In another preferred embodiment, the amino acid sequence of the antibody recognizing the tumor antigen has at least 90% identity with the amino acid sequences shown in any one of SEQ ID NO: 4 and SEQ ID NO: 14-22; preferably, the amino acid sequence of the antibody recognizing the tumor antigen is the amino acid sequence shown in any one of SEQ ID NO: 4 and SEQ ID NO: 14-22.

[0017] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor has at least 90% identity with the amino acid sequence shown in any one of SEQ ID NO: 23-44; preferably, the amino acid sequence of the chimeric antigen receptor is the amino acid sequence shown in any one of SEQ ID NO: 23-44.

[0018] In another preferred embodiment, the tumors include: breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvis cancer, spinal tumors, gliomas, pituitary adenomas, Kaposi's sarcoma, combinations of the cancers, and metastatic lesions of the cancers.

[0019] In another preferred embodiment, the tumors include: breast cancer, hematologic malignancies, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, and endocrine cancer. Systemic cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of the aforementioned cancers, and metastatic lesions of the aforementioned cancers.

[0020] In another preferred embodiment, the immune effector cells include: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, or bone marrow-derived phagocytes or combinations thereof; preferably, the immune effector cells are selected from autologous T cells, allogeneic T cells, or allogeneic NK cells, and more preferably, the T cells are autologous T cells.

[0021] In another preferred embodiment, gemcitabine is administered orally, intraperitoneally, and / or by injection.

[0022] In another preferred embodiment, lymphocyte removal is not performed on the individuals described.

[0023] A third aspect of the invention provides the use of immune effector cells expressing receptors that recognize tumor antigens in the preparation of a medicament, characterized in that the medicament contains the cells and gemcitabine for treating tumors or reducing tumor cell growth, survival, or viability in human patients, wherein the cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the effects of the cells and gemcitabine used alone.

[0024] A fourth aspect of the invention provides the use of immune effector cells expressing receptors that recognize tumor antigens and gemcitabine in the preparation of a medicament, characterized in that the medicament is used to treat tumors or reduce tumor cell growth, survival, or viability in human patients, wherein the cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the effects of the cells and gemcitabine used alone.

[0025] In a preferred embodiment, the immune effector cells are CAR T cells. Preferably, the CAR T cells recognize the epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin18.2, Claudin18.1, Claudin 6, phosphatidylinositol proteoglycan-3 (GPC3), BCMA, and / or vascular endothelial growth factor receptor.

[0026] A fifth aspect of the present invention provides a kit for treating tumors, the kit comprising:

[0027] 1) Immune effector cells that express receptors that recognize tumor antigens;

[0028] 2) Gesitabine;

[0029] 3) A container for containing the substances described in 1) and 2) above; and

[0030] 4) Instructions for use in treating tumors using the kit;

[0031] The immune effector cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the individual effects of the reagents; preferably, the immune effector cells are CAR T cells, more preferably, the CAR T cells recognize the epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin18.2, Claudin18.1, Claudin 6, phosphatidylinositol proteoglycan-3 (GPC3), BCMA and / or vascular endothelial growth factor receptor.

[0032] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0033] The beneficial effects of this invention are:

[0034] 1. The combination of gemcitabine and immune effector cells provided by this invention can significantly enhance the ability to kill tumor cells.

[0035] 2. The treatment regimen of this invention can counteract immunosuppression in the cancer microenvironment, thereby significantly enhancing its effect on solid tumors and showing good efficacy for refractory and progressive cancers. Attached Figure Description

[0036] Figure 1 is Image of the recombinant vector MSCV-8E5-2I-mBBZ plasmid.

[0037] Figure 2 shows This study demonstrated the inhibitory effect of gemcitabine combined with CAR-T cells on orthotopic pancreatic cancer in mice in vivo.

[0038] Figure 3 shows This study presents in vivo imaging data of mice treated with a combination of gemcitabine and CAR-T cells.

[0039] Figure 4 shows The study presented survival data for mice treated with a combination of gemcitabine and CAR-T cells. Detailed Implementation

[0040] This invention relates to the combined use of immune effector cells and gemcitabine in the treatment of tumors. It should be understood that this invention is not limited to the methods and experimental conditions described herein. Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the fields of gene therapy, biochemistry, genetics, molecular biology, and medicinal chemistry.

[0041] Similar or equivalent methods and materials described herein may be used in the practice or testing of this invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail. Furthermore, unless otherwise stated, materials, methods, and examples are illustrative only and not intended to be limiting.

[0042] Unless otherwise stated, the practice of this invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which fall within the scope of this art. These techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic AcidHybridization (BD Harries&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, APractical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols.154 and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MPCaloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volumes I-IV (DMWeir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0043] This invention is at least partly derived from the understanding that a combination of treatment regimens in which gemcitabine and immune effector cells are administered sequentially or substantially simultaneously for one or more cycles and / or doses can more effectively increase, enhance or prolong the activity and / or number of immune cells, thereby achieving an antitumor effect, in the treatment of cancer in some subjects.

[0044] The term "gemcitabine" has the chemical name 2'-deoxy-2',2'-difluorocytidine hydrochloride and the molecular formula C9H. 11 F2N3O4.HCl, with a molecular weight of 299.66. Gemcitabine is a pyrimidine antimetabolite antitumor drug that interferes with the synthesis and repair of tumor cell DNA by inhibiting ribonucleotide reductase and cell replication, and is effective against various solid tumors. In specific embodiments, the combination of gemcitabine and immunotherapy targeting tumor antigens can significantly enhance the antitumor effect, and even achieve complete remission.

[0045] The applicant also discovered that the present invention can not only improve the anti-cancer effect of refractory cancers, but also eliminate the need for lymphocyte clearance when using CAR-T cells, thereby greatly reducing the low anti-cancer treatment effect caused by lymphocyte clearance and reducing the toxic side effects caused by damage to normal tissues, especially the severe suppression of bone marrow.

[0046] In some implementations, the dose of gemcitabine administered intravenously to an individual with a tumor is approximately 1250 mg / m² per administration. 21250, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 mg / m² 2 In some implementations, individuals with tumors receive intravenous infusions of gemcitabine approximately every 6, 5, 4, 3, 2, or 1 week.

[0047] In this invention, the timing of administration of immune effector cells and gemcitabine is not critical; gemcitabine can be administered first, followed by immune effector cells; they can be administered simultaneously; or immune effector cells can be administered first, followed by gemcitabine. In some embodiments, immune effector cell therapy is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, before gemcitabine administration. In some implementations, immune effector cell therapy is administered at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, following gemcitabine administration.

[0048] The term "immune effector cells" refers to cells that perform effector functions during an immune response. For example, these include immune cells that secrete cytokines and / or chemokines, kill microorganisms, secrete antibodies, and recognize or eliminate tumor cells. In some embodiments, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages (or bone marrow-derived phagocytes), and dendritic cells. Preferably, the T cells include autologous T cells, xenogeneic T cells, or allogeneic T cells, and the natural killer cells are allogeneic NK cells.

[0049] As used in this article, the term "immune effector function or immune effector response" refers to the function or response of immune effector cells, such as enhancing or promoting the immune attack of target cells. For example, immune effector function or response refers to the property of T cells or NK cells that promote the killing of target cells or inhibit the growth or proliferation of target cells.

[0050] The term "immune effector function" includes any function mediated by components of the immune system that can lead to inhibition of tumor growth and / or inhibition of tumorigenesis, including inhibition of tumor spread and metastasis. Preferably, the immune effector function kills tumor cells. Preferably, the immune effector function in this invention is antibody-mediated, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells carrying tumor-associated antigens (e.g., by antibody binding to surface antigens), inhibition of CD40L-mediated signal transduction (e.g., by antibody binding to CD40 receptors or CD40 ligands (CD40L)), and / or inhibition of the proliferation of cells carrying tumor-associated antigens, preferably ADCC and / or CDC. Thus, antibodies capable of mediating one or more immune effector functions are preferably capable of mediating the killing of tumor cells by inducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypic adhesion, and / or phagocytosis (preferably by inducing CDC-mediated lysis and / or ADCC-mediated lysis). Antibodies can also exert their effects simply by binding to tumor-associated antigens on the surface of cancer cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis by binding to them on the surface of tumor cells.

[0051] The terms “therapeutic effective amount,” “therapeuticly effective,” and “effective amount” are used interchangeably herein to refer to the amount of a compound, preparation, substance, or composition that effectively achieves a specific biological outcome, such as, but not limited to, an amount or dose sufficient to promote a T-cell response. When indicated as “immunologically effective amount,” “antitumor effective amount,” “tumor-suppressive effective amount,” or “therapeutic effective amount,” the precise amount of immune effector cells or therapeutic agents administered according to the present invention can be determined by a physician taking into account an individual’s age, weight, tumor size, degree of infection or metastasis, and the patient’s (subject’s) condition. An effective amount of immune effector cells refers to, but is not limited to, the number of immune effector cells that increase, enhance, or prolong the antitumor activity of immune effector cells; increase the number of antitumor immune effector cells or activated immune effector cells; promote the secretion of IFN-γ and TNFα; or induce tumor regression, tumor shrinkage, or tumor necrosis.

[0052] The term "no lymphocyte clearance" or "no lymphocyte removal" means not removing lymphocytes from the subject's body. This includes, but is not limited to, not administering lymphocyte-clearing agents, whole-body radiation therapy, or combinations thereof, or other means of reducing lymphocyte count; however, if, after administering lymphocyte-clearing agents, whole-body radiation therapy, or combinations thereof, or other means of reducing lymphocyte count, the lymphocyte clearance rate in the subject is less than 60%, it will also fall under the scope of "no lymphocyte clearance" in this application.

[0053] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a sequence of proteins or peptides. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, “chimeric receptor” refers to a fusion molecule formed by linking DNA fragments or corresponding cDNAs of proteins from different sources using recombination technology, including extracellular, transmembrane, and intracellular domains. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs), modified T-cell (antigen) receptors (TCRs), T-cell fusion proteins (TFPs), and T-cell antigen couplers (TACs).

[0054] As used herein, "chimeric antigen receptor" or "CAR" refers to a group of peptides that, when present in immune effector cells, provide the cells with specificity for target cells (typically cancer cells) and exhibit intracellular signaling. A CAR typically comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes functional signaling domains derived from stimulatory and / or costimulatory molecules as defined below. In some respects, the peptide group is adjacent to each other. The peptide group includes dimerization switches that allow the peptides to couple to each other in the presence of dimerizing molecules, for example, that can couple the antigen-binding domain to the intracellular signaling domain. In one respect, the stimulatory molecule is a ζ-chain that binds to the T-cell receptor complex. In one respect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one respect, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, a CAR includes a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. In another aspect, a CAR includes a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In yet another aspect, a CAR includes a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and two functional signaling domains derived from one or more co-stimulatory molecules.

[0055] In one aspect, the present invention contemplates modifications to the amino acid sequence of an initiating antibody or fragment (e.g., scFv) to produce functionally equivalent molecules. For example, the VH or VL of the antigen-binding domain of a cancer-associated antigen described herein, such as the scFv contained in a CAR, can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity of the initiating VH or VL framework region (e.g., scFv) of the antigen-binding domain of the cancer-associated antigen described herein. The present invention contemplates modifications to the entire CAR construct, such as modifications to one or more amino acid sequences of multiple domains of the CAR construct, to produce functionally equivalent molecules. CAR constructs can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the starting CAR construct.

[0056] In one specific embodiment, the amino acid sequence of the antibody for recognizing tumor antigens herein has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4, or as shown in SEQ ID NO: 14, or as shown in SEQ ID NO: 15, or as shown in SEQ ID NO: 16, or as shown in SEQ ID NO: 17, or as shown in SEQ ID NO: 18, or as shown in SEQ ID NO: 19, or as shown in SEQ ID NO: 20, or as shown in SEQ ID NO: 21, or as shown in SEQ ID NO: 22; preferably, the amino acid sequence shown in SEQ ID NO: 4, or as shown in SEQ ID NO: 14, or as shown in SEQ ID NO: 15, or as shown in SEQ ID NO: 16, or as shown in SEQ ID NO: 17, or as shown in SEQ ID NO: 18, or as shown in SEQ ID NO: 19, or as shown in SEQ ID NO: 20, or as shown in SEQ ID NO: 21, or as shown in SEQ ID NO: 22.

[0057] As used herein, a "transmembrane domain" refers to a region of a protein sequence that crosses the cell membrane. This may include one or more additional amino acids adjacent to the transmembrane domain, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids in the extracellular region) and / or one or more additional amino acids associated with the extracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids in the intracellular region). In one aspect, a transmembrane domain is a domain associated with one of the other domains of a chimeric receptor; for example, in one embodiment, the transmembrane domain may originate from the same protein from which a signal transduction domain, co-stimulatory domain, or hinge domain originates. In some cases, a transmembrane domain may be selectively or modified by amino acid substitution to prevent such a domain from binding to transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of isodimerizing with another chimeric receptor on the cell surface expressing the chimeric receptor. The transmembrane domain can be derived from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the chimeric receptor binds to the target antigen. The transmembrane domain particularly used in this invention may include at least the following transmembrane domains: for example, the α, β, or ζ chain of the T-cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.In some embodiments, the transmembrane domain may include at least the following transmembrane regions: for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C.

[0058] In some cases, the transmembrane domain can be linked to the extracellular region of the CAR, such as the antigen-binding domain, via a hinge (e.g., a hinge derived from human proteins). For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., the GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In one aspect, the transmembrane domain can be recombinant, in which case it will primarily contain hydrophobic residues, such as leucine and valine. In another aspect, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain. Optionally, short oligopeptide or polypeptide linkers with a length between 2 and 10 amino acids can form a bond between the transmembrane domain of the CAR and the cytoplasmic region. A glycine-serine dinucleotide provides a particularly suitable linker.

[0059] The term "intracellular domain" or "cytoplasmic domain" as used herein includes intracellular signal transduction domains. Intracellular signal transduction domains are typically responsible for the activation of at least one of the normal immune effector functions of immune cells in which chimeric receptors have been introduced. The term "effector function" refers to a specialized function of the cell. Immune effector functions of T cells can be, for example, cytolytic or cofactor activities, including the secretion of cytokines. Therefore, the term "intracellular signal transduction domain" refers to a portion of a protein that transduces immune effector function signals and directs the cell to perform a specific function. While the entire intracellular signal transduction domain can generally be used, in many cases it is not necessary to use the entire chain. Regarding the use of truncated portions of intracellular signal transduction domains, such truncated portions can be used instead of the complete chain, provided they transduce immune effector function signals. Therefore, the term intracellular signal transduction domain means a truncated portion of an intracellular signal transduction domain that is sufficient to transduce immune effector function signals.

[0060] It is well known that the signals generated by the TCR alone are insufficient to fully activate T cells, and secondary and / or co-stimulatory signals are also required. Therefore, T cell activation can be described as being mediated by two different types of cytoplasmic signaling sequences: those that induce antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, such as co-stimulatory domains).

[0061] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that stimulates the activation of immune cells in at least some aspects of immune cell signaling pathways. In one aspect, the signal is a primary signal initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC-antigen peptide complex, and it leads to a T cell-mediated response, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence acting in a stimulatory manner (also referred to as a "primary signaling domain") may contain a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing cytoplasmic signaling sequences particularly used in this invention include, but are not limited to, those derived from: CD3ζ, the common FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. The intracellular signal transduction domain in any CAR of the present invention includes an intracellular signal transduction sequence, such as the primary signal transduction sequence of CD3-ζ. In the CAR of the present invention, the primary signal transduction sequence of CD3-ζ is an equivalent residue derived from human or non-human species such as mice, rodents, monkeys, apes, etc.

[0062] The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response in the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that promote an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, and LFA. -1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0063] Costimulatory intracellular signal transduction domains can be the intracellular portion of costimulatory molecules. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte-activating molecules (SLAM proteins), and NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAM-MF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.

[0064] Intracellular signal transduction domains can include all intracellular portions of a molecule or all of its natural intracellular signal transduction domains, or functional fragments or derivatives thereof.

[0065] The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided in GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.; and the "4-1BB co-stimulatory domain" is defined as amino acid residues 214–255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. In one aspect, the "4-1BB co-stimulatory domain" is an equivalent residue from humans or from non-human species such as mice, rodents, monkeys, apes, etc.

[0066] The chimeric antigen receptor described in this article has: (i) an antibody or fragment thereof that recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody or fragment thereof that recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and CD3ζ; or (iii) an antibody or fragment thereof that recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of CD137, and CD3ζ.

[0067] The amino acid sequence of the chimeric antigen receptor described herein has at least 90% identity with the amino acid sequences shown in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44; preferably, the amino acid sequence of the chimeric antigen receptor is any of the amino acid sequences shown in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44.

[0068] The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0069] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., through binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, scFv, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies (such as sdAb), camelid VHH domains, multispecific antibodies formed from antibody fragments (e.g., bivalent fragments comprising two Fab fragments linked by disulfide bonds in a hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short, flexible polypeptide linker) and are capable of being expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv may include VL-connector-VH or may include VH-connector-VL.

[0070] The term "antibody heavy chain" refers to the larger of two polypeptide chains that are present in the antibody molecule in their naturally occurring configuration and usually determine the type of antibody.

[0071] The term "antibody light chain" refers to the smaller of two polypeptide chains that exist in the antibody molecule in their naturally occurring configuration. κ(k) and λ(1) light chains refer to the isotypes of the two main antibody light chains.

[0072] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as antibodies expressed by phage or yeast expression systems. This term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding an antibody (wherein the DNA molecule expresses an antibody protein) or specifying the amino acid sequence of an antibody, wherein said DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequencing techniques available and well-known in the art.

[0073] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of cells with specific immune capabilities, or both. Those skilled in the art will understand, and indeed, that any macromolecule, whether a protein or peptide, can act as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. When used herein, those skilled in the art will understand that this includes any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily need to be encoded solely by the full-length nucleotide sequence of a gene. This invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode a polypeptide that elicits the desired immune response. Those skilled in the art will understand that antigens do not necessarily need to be encoded by a "gene". Antigens can be synthesized, or can be derived from a biological sample, or can be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids containing other biological components.

[0074] The term "tumor-associated antigen (TAA)" or "tumor antigen" refers to a protein that is specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific developmental stage. In some embodiments, the tumor-associated antigen is specifically expressed in gastric tissue under normal conditions, preferably in the pancreas, gastric mucosa, reproductive organs (e.g., testes), trophoblastic tissue (e.g., placenta), or germline cells, and expressed or abnormally expressed in one or more tumor tissues or cancerous tissues. In some embodiments, the tumor-associated antigen refers to a differentiation antigen, preferably a cell type-specific differentiation antigen, i.e., a protein specifically expressed in a specific cell type at a specific differentiation stage under normal conditions. In some embodiments, Tumor-associated antigens (TAs) are located on tumor cells but are not expressed or are expressed in small amounts in normal tissues. In some embodiments, TAs or anomalous expression of TAs can be used to identify tumor cells. In some embodiments, the TAs expressed by tumor cells in an individual (e.g., a cancer patient) are autologous proteins of that individual. In some embodiments, TAs are specifically expressed in tissues or organs that are not lethal to the individual under normal conditions when the individual is damaged by the immune system; or in organs or structures that are inaccessible or difficult for the body's immune system to access. In some embodiments, the amino acid sequence of TAs expressed in normal tissues is identical to that of TAs expressed in tumor tissues.

[0075] The tumor antigens of this invention include, but are not limited to: thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin-13 receptor subunit α (IL-13Rα); Interleukin-11 receptor α (IL-11Rα); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor, Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryonic antigen-4 (SSEA-4); Cell surface-associated mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); LMP2; Hepatic glycoside A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3; TGS5; High molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside; Folic acid receptor; Tumor vascular endothelial marker 1 (TEM1 / CD248); Tumor vascular endothelial marker 7-associated (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; κ light chain (kappa) lightchain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tendinin; carcinoembryonic variant of tumor necrosis region; G protein-coupled receptor class C5-member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH hexose moiety of glycoceramide (GloboH);Breast differentiation antigen (NY-BR-1); uroplakin2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenaline receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); nephroblastoma protein (WT1); ETS translocation variant gene 6 (ETV6-AML); Sperminin 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 ( MAD-CT-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (Rho C); Cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); Squamous cell carcinoma antigen 3 recognized by T cells (SART3); Pairing box protein Pax-5 (PAX5); Proacrosin-binding protein sp32 (OYTES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); Synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAI) R1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLL1).

[0076] The term "tumor" refers to an expansion or lesion caused by abnormal cell growth (called neoplastic cells or tumor cells). "Tumor cells" are abnormal cells that proliferate rapidly and uncontrollably, continuing to grow even after the stimulus causing this proliferation has ceased. Tumors are characterized by a partial or complete lack of structural tissue and functional coordination with normal tissue, typically forming a distinctive mass of tissue that can be benign, pre-malignant, or malignant.

[0077] The term "cancer" (medical term: malignancy) is a group of diseases in which a group of cells exhibits uncontrolled growth (dividing beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasis (spreading to other locations in the body via the lymphatic system or bloodstream). These three malignant characteristics of cancer distinguish it from benign tumors, which are self-limiting and do not invade or metastasize. Most cancers form tumors, but some (such as leukemia) do not.

[0078] The terms "cancer" and "tumor," or "cancer disease" and "tumor disease," are used interchangeably to refer to diseases in which cells exhibit uncontrolled growth and / or invasion and / or metastasis. This includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancers, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose, and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as their metastases. Examples include lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases from any of the above-mentioned cancers or tumors.

[0079] The terms “transfection” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0080] The terms “specifically bind” and “specifically recognize” have the same meaning in this article, referring to an antibody or ligand that recognizes and binds to an antigen (e.g., a tumor antigen) present in a sample, but which does not substantially recognize or bind to other molecules in the sample.

[0081] The term "refractory" refers to a disease, such as cancer or tumor, that does not respond to treatment. In some embodiments, a refractory tumor may be resistant to treatment prior to or at the start of treatment. In other embodiments, a refractory tumor may be resistant during treatment. Refractory cancer is also referred to as a resistant tumor. In this invention, refractory tumors include, but are not limited to, tumors that are insensitive to radiotherapy, relapse after radiotherapy, are insensitive to chemotherapy, relapse after chemotherapy, are insensitive to CAR-T therapy, or relapse after treatment. Refractory or recurrent malignant tumors can be treated using the treatment regimens described herein.

[0082] As used in this article, “relapsed” means the return of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement, for example, after prior treatment with a therapy, such as a cancer therapy.

[0083] The terms "object," "individual," "organism," or "patient" are used interchangeably, including vertebrates, preferably mammals. In this invention, mammals include humans, non-human primates, domesticated animals (e.g., dogs, cats, sheep, cattle, goats, pigs, horses, etc.), laboratory animals (e.g., mice, rats, rabbits, guinea pigs, etc.), and captive animals (e.g., animals in zoos). The term "animal" as used herein also includes humans. The terms "object" and "individual" include patient, i.e., a diseased animal, preferably a human, where the disease is preferably one described herein.

[0084] The term “enhancement” refers to the ability of a subject or tumor cells to improve their response to a treatment disclosed herein. For example, an enhanced response may include an increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more in responsiveness. As used herein, “enhancement” may also refer to an increase in the number of subjects responding to a treatment, such as an immune effector cell therapy. For example, an enhanced response may refer to the total percentage of subjects responding to treatment, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more.

[0085] In one respect, treatment is determined by clinical outcomes; through increased, enhanced, or prolonged anti-tumor activity of T cells; an increase in the number of anti-tumor T cells or activated T cells compared to pre-treatment levels; promotion of IFN-γ, TNFα secretion, or a combination thereof. In another respect, clinical outcomes include tumor regression; tumor shrinkage; tumor necrosis; an anti-tumor response of the immune system; tumor enlargement, recurrence, or metastasis, or a combination thereof. In yet another respect, the therapeutic effect is predicted by the presence of T cells, the presence of gene markers indicating T-cell inflammation, promotion of IFN-γ, TNFα secretion, or a combination thereof.

[0086] As disclosed herein, immune effector cells can be administered to individuals via various routes, including, for example, oral or extra-gastric, intravenous, intramuscular, subcutaneous, intraorbital, intracystic, intraperitoneal, intrarectal, intracisional, intratumoral, intranasal, intradermal, or passive or enhanced absorption through the skin via, for example, skin patches or transdermal iontophoresis.

[0087] In practicing the methods of this invention, the total amount of reagent to be administered can be given to the subject as a single dose via bolus injection or by infusion over a relatively short period of time, or it can be administered using a tiered treatment regimen in which multiple doses are administered over an extended period of time. Those skilled in the art will know that the amount of composition used to treat a pathological condition in a subject depends on many factors, including the subject's age and general health, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, those skilled in the art will adjust the specific dosage as needed. Generally, initially, the formulation of the composition and the route and frequency of administration are determined using Phase I and Phase II clinical trials.

[0088] Scope: Throughout this disclosure, all aspects of the invention may be expressed in the form of a scope. It should be understood that the scope description is for convenience and brevity only and should not be construed as an immutable limitation on the scope of the invention. Therefore, the scope description should be considered to specifically disclose all possible sub-scopes and individual values ​​within that scope. For example, a scope description from 1 to 6 should be considered to specifically disclose sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual values ​​within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope of 95-99% identity includes scopes having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the scope.

[0089] Based on this disclosure, those skilled in the art will understand that many variations or modifications can be made to the specific embodiments disclosed, while still obtaining the same or similar results without departing from the spirit and scope of the invention. The invention is not limited in scope to the specific embodiments described herein (which are intended only as illustrative examples of various aspects of the invention), and functionally equivalent methods and components are within the scope of the invention.

[0090] This application relates to adoptive or immune effector cell therapy for solid tumors, including administering multiple or repeated doses of cells, and methods, compositions, and articles thereof. The cells generally express chimeric antigen receptors, such as chimeric antigen receptors (CARs) or other transgenic receptors such as T-cell receptors (TCRs).

[0091] Human claudin 18 (CLD18) molecules (Genbank registry numbers: splice variant 1 (CLD18A1 or Claudin 18.1): NP_057453, NM016369, and splice variant 2 (CLD18A2 or Claudin 18.2): NM_001002026, NP_001002026) are intrinsic transmembrane proteins with a molecular weight of approximately 27.9 / 27.72 kDa. Mouse claudin 18.2 (NP_001181850.1).

[0092] The term "CLD18" refers to tight protein 18 and includes any CLD18 variants (including CLD18A1 and CLD18A2), conformations, isoforms, and species homologs expressed in cells naturally expressed or in cells transfected with the CLD18 gene. Tight protein 18 is an intrinsic membrane protein located in tight junctions between epithelial and endothelial cells. Tight junctions form a network of interconnected granular chains within the tissue membrane between adjacent cells. In tight junctions, occludin and tight protein are the most prevalent transmembrane protein components. Due to their strong intercellular adhesion properties, they create a primary barrier that prevents and controls paracellular transport of solutes and restricts lateral diffusion of membrane lipids and proteins to maintain cell polarity. Proteins forming tight junctions are crucially involved in the structure of tissue epithelial tissues. Preferably, “CLD18” refers to human CLD18, particularly CLD18A2 (amino acid sequence as shown in SEQ ID NO: 2) and / or CLD18A1 (amino acid sequence as shown in SEQ ID NO: 3), more preferably CLD18A2.

[0093] The term “CLD18A1” includes post-translational variants, isoforms, and interspecies homologs of CLD18A1 expressed by cells that are naturally expressed or transfected with the CLD18A1 gene.

[0094] The term “CLD18A2” includes post-translational variants, isoforms, and interspecies homologs of CLD18A2 expressed in cells that are naturally expressed or transfected with the CLD18A2 gene.

[0095] The term “CLD18 variant” should include (i) CLD18 splice variants, (ii) CLD18 post-translational modification variants, particularly variants with different N-glycosylation states, (iii) CLD18 conformation variants, particularly CLD18-conformation-1, CLD18-conformation-2 and CLD18-conformation-3, (iv) free CLD18 and isotype / heterotype-associated variants located at tight junctions, and (v) CLD18 cancer-associated variants and CLD18 non-cancer-associated variants.

[0096] In some embodiments, the dense protein 18A2 is a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant of the amino acid sequence. The term "variant" refers to mutants, splice variants, conformations, isotypes, allele variants, species variants, and species homologs, particularly naturally occurring variants. The terms "CLDN," "CLDN18," "CLDN18A1," and "CLDN18A2" should cover any post-translational modification variants and conformational variants.

[0097] CLD18A1 is selectively expressed in the epithelium of normal lungs and stomach, while CLD18A2 is expressed only in gastric cells. Furthermore, CLD18A2 is confined to differentiated short-lived gastric epithelial cells but is absent in gastric stem cell regions.

[0098] In some embodiments, the primary tumor refers to gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, and the metastasis specifically refers to gastric cancer metastases such as Krukenberg tumor, peritoneal metastases, and lymph node metastases. In some embodiments, cells expressing CLDN18A2 refer to tumor cells, and are particularly selected from tumorigenic gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, lung cancer cells, ovarian cancer cells, colon cancer cells, liver cancer cells, head and neck cancer cells, and gallbladder cancer cells.

[0099] In some embodiments, the tumors include: breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvis cancer, spinal tumors, gliomas, pituitary adenomas, Kaposi's sarcoma, combinations of the above cancers, and metastatic lesions of the above cancers.

[0100] The present invention provides a treatment method and composition for treating diseases (e.g., tumors) with CLD18 expression.

[0101] This invention provides a method for treating a subject's tumor using adoptive or immune effector cell therapy for solid tumors, wherein the subject expresses a genetically engineered (recombinant) chimeric immune effector cell receptor. The method generally includes single-cycle or multiple-cycle infusion of such cells. In some embodiments, this involves dose escalation within a cycle, repeated dose infusions within a cycle, or dose reduction within a cycle. In some embodiments, the initial dose is a lower dose and / or a moderating or tapering dose, and / or subsequent doses are consolidation doses and / or moderating or tapering doses. Cells, compositions, and articles for use in such methods are also provided. In some embodiments, the chimeric receptor is a genetically engineered antigen receptor, such as a functional non-TCR antigen receptor, for example, a chimeric antigen receptor (CAR), and other recombinant antigen receptors such as transgenic T-cell receptors (TCRs). Receptors also include other recombinant chimeric receptors, such as those containing extracellular and intracellular portions that specifically bind to a ligand or receptor or other binding partner, such as the intracellular signal transduction portion of a CAR. In some embodiments, the dose includes a lower initial dose.

[0102] In some embodiments, the method includes (a) administering a subject with a tumor a single course of cells expressing a chimeric antigen receptor (e.g., CAR); and (b) administering the subject multiple courses of cells expressing a chimeric antigen receptor (e.g., CAR). In other embodiments, one or more subsequent doses may be administered.

[0103] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., *Molecular Cloning: A Laboratory Manual (Third Edition)* (Science Press, 2002), or according to the manufacturer's recommendations.

[0104] The exemplary antigen receptors of the present invention include CARs, and methods for engineering and introducing the receptors into cells, refer to, for example, those disclosed in Chinese Patent Application Publications CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, and International Patent Application Publications WO2017186121A1, WO2018006882A1, WO2015172339A8, and WO2018 / 018958A1.

[0105] Example 1: Establishment of Panc02 / Luc-GFP-Claudin 18.2, 8E5-2I-mBBZ CAR T cells

[0106] 1. Construction of Panc02 / Luc-GFP-Claudin 18.2 cells

[0107] A Panc02 pancreatic cancer cell model overexpressing mouse Claudin 18.2 (SEQ ID NO: 1) was established using conventional molecular biology techniques (the Panc02 pancreatic cancer cell model was purchased from Beina Biotechnology). Using pWPT as a vector, mouse Claudin 18.2 (SEQ ID NO: 1) was inserted to construct the plasmid pWPT-mClaudin 18.2. Then, a lentiviral packaging infection method was used to establish Panc02 / Luc-GFP-Claudin 18.2 pancreatic cancer cells overexpressing mouse Claudin 18.2.

[0108] 2. Construction of T cells expressing chimeric antigen receptors

[0109] (1) Plasmid construction

[0110] Using conventional molecular biology methods in this field, the scFv used in this embodiment is an antibody targeting mouse claudin18.2, and its nucleic acid sequence is shown in SEQ ID NO: 5.

[0111] Using MSCV-IRES-GFP (purchased from Addgene) as a vector, a retroviral plasmid expressing a second-generation chimeric antigen receptor, MSCV-8E5-2I-mBBZ, was constructed. Figure 1(See diagram). The 8E5-2I-mBBZ sequence consists of a mouse CD8α signal peptide (the amino acid sequence of which is shown in SEQ ID NO: 6 and the nucleotide sequence of which is shown in SEQ ID NO: 7), a claudin18.2-targeting scFv (the amino acid sequence of which is shown in SEQ ID NO: 4 and the nucleotide sequence of which is shown in SEQ ID NO: 5), a mouse CD8 hinge and transmembrane region (the amino acid sequence of which is shown in SEQ ID NO: 8 and the nucleotide sequence of which is shown in SEQ ID NO: 9), a mouse 4-1BB intracellular signal transduction domain (the amino acid sequence of which is shown in SEQ ID NO: 10 and the nucleotide sequence of which is shown in SEQ ID NO: 11), and an intracellular segment of mouse CD3, CD3ζ (the amino acid sequence of which is shown in SEQ ID NO: 12 and the nucleotide sequence of which is shown in SEQ ID NO: 13).

[0112] The constructed MSCV-8E5-2I-mBBZ was transfected into 293T cells (derived from ATCC) to package retroviruses, yielding retroviruses. The infection method was a routine method used in the preparation of T cells expressing chimeric antigen receptors in this field.

[0113] (2) CAR T cell construction: Splenic T lymphocytes from C57BL / 6 mice (from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.) were collected. The purified mouse CD3+ T lymphocytes were added to Dynabeads MouseT-activator CD3 / CD28 at a volume ratio of 1:1. After washing once with PBS, the cells were activated and cultured in an incubator. The culture medium was RPMI 1640 complete medium (purchased from Invitrogen) with 10% FBS serum added.

[0114] Mouse spleen T lymphocytes activated for 24 hours were seeded into 12-well plates coated with recombinant human fibrin fragments, and infected with retroviruses for 12 hours. The cells were then cultured and expanded to the required number to obtain mouse 8E5-2I-mBBZ CAR T cells.

[0115] Example 2: In vivo experimental observation of the inhibitory effect of gemcitabine combined with CAR T cells on pancreatic cancer in mice.

[0116] 2 × 10⁶ pancreas cells were orthotopically injected into 6-week-old C57BL / 6 mice. 5 Panc02 / Luc-GFP-Claudin 18.2 cells, tumor cell inoculation diary day 0.

[0117] On day 6 post-tumor inoculation, mouse T cells were harvested and the 8E5-2I-mBBZ CART cell line was constructed according to Example 1. Simultaneously, in vivo imaging of the mice was performed, and mice were randomly divided into groups based on tumor burden. The primary group was:

[0118] Group 1: UTD group, which is the group that was treated with only untransfected mouse T cells;

[0119] Group 2: CAR-T group, which is the group that received only 8E5-2I-mBBZ CAR T-cell therapy;

[0120] Group 3: Gemcitabine group (or gemcitabine group), i.e., the group receiving gemcitabine monotherapy; and

[0121] Group 4: Gemcitabine+CART group (or gemcitabine+CART group), which is the group receiving 8E5-2I-mBBZCAR T cell therapy combined with gemcitabine.

[0122] On days 6, 7, and 8, mice in the Gemcitabine group and the Gemcitabine+CART group were treated with 30 mg / kg gemcitabine via intraperitoneal administration once daily.

[0123] On the 9th day after tumor inoculation (Day 9), mice in the CART group and Gemcitabine+CART group were given a tail vein injection of 3×1068E5-2I-mBBZ CAR-T cells, while the UTD group was injected with 3×106 untransfected mouse T cells. The treatment effect was then observed by in vivo imaging once a week.

[0124] The results of the inhibitory effect on orthotopic mouse pancreatic cancer are as follows: Figure 2 The results showed that on day 13 after treatment, compared with the UTD group, CART group, and Gemcitabine group, the tumor burden (highlighted in white) in the Gemcitabine+CART group was significantly reduced, and two mice in the Gemcitabine+CART group achieved complete remission. On day 21, four mice in the Gemcitabine+CART group achieved complete remission. In contrast, no mice in the other three groups achieved complete remission on day 21. In vivo imaging data showed that tumors continued to grow in the UTD, CART, and Gemcitabine groups; compared with the UTD group, tumor growth was effectively controlled in the Gemcitabine+CART group (P < 0.05). Figure 3)Mouse survival data showed that the Gemcitabine+CART group had the longest survival time, significantly exceeding that of the UTD group (P<0.01). Figure 4) and the survival time of mice in the Gemcitabine group (P < 0.05, Figure 4) .

[0125] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0126] The sequences involved in this application are as follows:

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] sequence list <110> CARsgen Therapeutics (Shanghai) Co., Ltd. Shanghai Cancer Institute <120> Combination of cell immunotherapy <130> PE01624A-FD00194CN <141> 2020-01-07 <150> 201910012164.9 <151> 2019-01-07 <160> 44 <170> SIPOSequenceListing 1.0 <210> 1 <211> 795 <212> DNA <213> Artificial Sequence <400> 1 atgtcggtga ccgcctgcca gggcttgggg tttgtggtgt cactgatcgg gtttgcgggc 60 atcattgcag ccacttgtat ggaccagtgg agcacccagg atttatacaa caacccggtg 120 accgctgtat tcaactacca agggctatgg cgttcatgcg tccgagagag ctctggcttc 180 accgagtgcc gaggctactt caccctgttg gggttgccag ccatgctgca agctgtacga 240 gccctgatga tcgtgggcat tgttctgggg gtcatcggta tcctcgtgtc catcttcgcc 300 ctgaagtgca ttcgcattgg tagcatggat gactctgcca aggccaagat gactctgact 360 tctgggatct tgttcatcat ctccggcatc tgtgcaatca ttggtgtgtc tgtgtttgcc 420 aacatgctgg tgaccaactt ctggatgtcc acagctaaca tgtacagcgg catgggcggc 480 atgggtggca tggtgcagac cgttcagacc aggtacacct ttggtgcagc tctgttcgtg 540 ggctgggttg ctggaggcct caccctgatt gggggagtga tgatgtgcat cgcctgccgt 600 ggcctgacac cagatgacag caacttcaaa gctgtgtctt accatgcctc tggccaaaat 660 gttgcctaca ggcctggagg ctttaaggcc agcactggct ttgggtccaa caccagaaac 720 aagaagatct acgatggggg tgcccgcaca gaagacgatg aacagtctca tcctaccaag 780 tatgactatg tgtag 821 <210> 2 <211> 261 <212> PRT <213> Artificial Sequence <400> 2 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 3 <211> 261 <212> PRT <213> Artificial Sequence <400> 3 Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 4 <211> 247 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Glue Ile Lys Arg 245 <210> 5 <211> 741 <212> DNA <213> Artificial Sequence <400> 5 caggtgcagc tgcaggag cggccccggc ctgatcaagc ccagccagac cctgagcctg 60 acctgcaccg tgagcggcgg cagcatcagc agcggctaca acctgcactg gatccggcag 120 ccccccggca agggcctgga gtggatcggc tacatccact acacggcag caccactac 180 aaccccgccc tgcggagccg ggtgaccatc agcgtggaca ccagcaagaa ccagttcagc 240 ctgaagctga gcagcgtgac cgccgccgac accccatct actactgcgc ccggatctac 300 aacggcaaca gcttccccta ctggggccag ggcaccaccg tgaccgtgag cagcggtgga 360 ggcggttcag gcggaggtgg ttctggcggt ggcggatcgg acatcgtgat gacccagagc 420 cccgacagcc tggccgtgag cctgggcgag cgggccacca tcaactgcaa gagcagccag 480 agcctgttca acagcggcaa ccagaagaac tacctgacct ggtaccagca gaagcccggc 540 cagcccccca agctgctgat ctactgggcc agcacccggg agagcggcgt gcccgaccgg 600 ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcaggccgag 660 gacgtggccg tgtactactg ccagaacgcc tacagcttcc cctacacctt cggcggcggc 720 accaagctgg agatcaagcg g 765 <210> 6 <211> 27 <212> PRT <213> Artificial Sequence <400> 6 Put Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu Assn Leu Leu Leu Leu 1 5 10 15 Gly Glu Ser Ile Ile Leu Gly Ser Gly Glu Ala 20 25 <210> 7 <211> 81 <212> DNA <213> Artificial Sequence <400> 7 atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60 atcctgggga gtggagaagc t 83 <210> 8 <211> 72 <212> PRT <213> Artificial Sequence <400> 8 Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr 1 5 10 15 Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser 20 25 30 Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 35 40 45 Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr 50 55 60 Leu Ile Cys Tyr His Arg Ser Arg 65 70 <210> 9 <211> 216 <212> DNA <213> Artificial Sequence <400> 9 actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60 ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120 gcctgtgata tttacatctg ggcacccttg gccggaatct gcgtggccct tctgctgtcc 180 ttgatcatca ctctcatctg ctaccacagg agccga 222 <210> 10 <211> 45 <212> PRT <213> Artificial Sequence <400> 10 Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys 1 5 10 15 Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys 20 25 30 Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu 35 40 45 <210> 11 <211> 135 <212> DNA <213> Artificial Sequence <400> 11 aaatggatca ggaaaaaatt cccccacata ttcaagcaac catttaagaa gaccactgga 60 gcagctcaag aggaagatgc ttgtagctgc cgatgtccac aggaagaaga aggaggagga 120 ggaggctatg agctg 139 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <400> 12 Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp Pro Asn Gln Leu 1 5 10 15 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Glu 20 25 30 Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys Gln Gln Arg Arg 35 40 45 Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln Lys Asp Lys Met 50 55 60 Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu Arg Arg Arg Gly 65 70 75 80 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 85 90 95 Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala 100 105 <210> 13 <211> 321 <212> DNA <213> Artificial Sequence <400> 13 agcaggagtg cagagactgc tgccaacctg caggacccca accagctcta caatgagctc 60 aatctagggc gaagagagga atatgacgtc ttggagaaga agcgggctcg ggatccagag 120 atgggaggca aacagcagag gaggaggac ccccaggaag gcgtatacaa tgcactgcag 180 aaagacaaga tggcagaagc ctacagtgag atcggcaca aaggcgagag gcggagaggc 240 aaggggcacg atggccttta ccagggctctc agcactgcca ccaggacac ctatgatgcc 300 ctgcatatgc agaccctggc c 331 <210> 14 <211> 247 <212> PRT <213> Artificial Sequence <400> 14 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg 245 <210> 15 <211> 242 <212> PRT <213> Artificial Sequence <400> 15 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Tyr Arg Tyr Asp Glu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala 130 135 140 Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Ile Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Thr 165 170 175 Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 195 200 205 Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln 210 215 220 Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Arg <210> 16 <211> 247 <212> PRT <213> Artificial Sequence <400> 16 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Phe Ser Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Ser Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg 245 <210> 17 <211> 247 <212> PRT <213> Artificial Sequence <400> 17 Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Met Glu Trp 35 40 45 Met Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Arg Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Asp Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Pro Gly Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Arg Pro Gly Gln Pro Pro Lys Met Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Phe Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg 245 <210> 18 <211> 247 <212> PRT <213> Artificial Sequence <400> 18 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg 245 <210> 19 <211> 247 <212> PRT <213> Artificial Sequence <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg 245 <210> 20 <211> 247 <212> PRT <213> Artificial Sequence <400> 20 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg 245 <210> 21 <211> 247 <212> PRT <213> Artificial Sequence <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Val Glu Ile Lys Arg 245 <210> 22 <211> 247 <212> PRT <213> Artificial Sequence <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg 245 <210> 23 <211> 465 <212> PRT <213> Artificial Sequence <400> 23 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr 130 135 140 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val 145 150 155 160 Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 165 170 175 Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg 180 185 190 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn 195 200 205 Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn 210 215 220 Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 225 230 235 240 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 245 250 255 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 260 265 270 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 275 280 285 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 290 295 300 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 305 310 315 320 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 325 330 335 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 340 345 350 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 355 360 365 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 370 375 380 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 385 390 395 400 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 405 410 415 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 420 425 430 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 435 440 445 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 450 455 460 Arg 465 <210> 24 <211> 462 <212> PRT <213> Artificial Sequence <400> 24 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr 130 135 140 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val 145 150 155 160 Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 165 170 175 Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg 180 185 190 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn 195 200 205 Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn 210 215 220 Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 225 230 235 240 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 245 250 255 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 260 265 270 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 275 280 285 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 290 295 300 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 305 310 315 320 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 325 330 335 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 340 345 350 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn 355 360 365 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 370 375 380 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 385 390 395 400 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 405 410 415 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 420 425 430 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 435 440 445 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 <210> 25 <211> 507 <212> PRT <213> Artificial Sequence <400> 25 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr 130 135 140 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val 145 150 155 160 Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 165 170 175 Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg 180 185 190 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn 195 200 205 Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn 210 215 220 Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 225 230 235 240 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 245 250 255 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 260 265 270 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 275 280 285 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 290 295 300 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 305 310 315 320 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 325 330 335 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 340 345 350 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 355 360 365 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 370 375 380 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 385 390 395 400 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 405 410 415 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 420 425 430 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn 435 440 445 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 450 455 460 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 465 470 475 480 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 485 490 495 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 <210> 26 <211> 473 <212> PRT <213> Artificial Sequence <400> 26 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln 405 410 415 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 27 <211> 470 <212> PRT <213> Artificial Sequence <400> 27 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460 Gln Ala Leu Pro Pro Arg 465 470 <210> 28 <211> 515 <212> PRT <213> Artificial Sequence <400> 28 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 355 360 365 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 370 375 380 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 385 390 395 400 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 405 410 415 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 420 425 430 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 435 440 445 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 450 455 460 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 465 470 475 480 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 485 490 495 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 500 505 510 Pro Pro Arg 515 <210> 29 <211> 472 <212> PRT <213> Artificial Sequence <400> 29 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 245 250 255 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 260 265 270 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 275 280 285 Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 290 295 300 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 305 310 315 320 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 325 330 335 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 340 345 350 Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp 355 360 365 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 370 375 380 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 385 390 395 400 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 405 410 415 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 420 425 430 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 435 440 445 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 450 455 460 His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 30 <211> 469 <212> PRT <213> Artificial Sequence <400> 30 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 245 250 255 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 260 265 270 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 275 280 285 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 290 295 300 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 305 310 315 320 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 325 330 335 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 340 345 350 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 355 360 365 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 370 375 380 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 385 390 395 400 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 405 410 415 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 420 425 430 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 435 440 445 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 450 455 460 Ala Leu Pro Pro Arg 465 <210> 31 <211> 514 <212> PRT <213> Artificial Sequence <400> 31 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 245 250 255 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 260 265 270 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 275 280 285 Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 290 295 300 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 305 310 315 320 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 325 330 335 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 340 345 350 Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr 355 360 365 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 370 375 380 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 385 390 395 400 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 405 410 415 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 420 425 430 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 435 440 445 Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 450 455 460 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 465 470 475 480 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 485 490 495 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 500 505 510 Pro Arg <210> 32 <211> 473 <212> PRT <213> Artificial Sequence <400> 32 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln 405 410 415 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 33 <211> 470 <212> PRT <213> Artificial Sequence <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460 Gln Ala Leu Pro Pro Arg 465 470 <210> 34 <211> 515 <212> PRT <213> Artificial Sequence <400> 34 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr Lys Leu Glu Leu Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 355 360 365 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 370 375 380 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 385 390 395 400 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 405 410 415 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 420 425 430 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 435 440 445 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 450 455 460 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 465 470 475 480 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 485 490 495 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 500 505 510 Pro Pro Arg 515 <210> 35 <211> 473 <212> PRT <213> Artificial Sequence <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Val Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln 405 410 415 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 36 <211> 470 <212> PRT <213> Artificial Sequence <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Val Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460 Gln Ala Leu Pro Pro Arg 465 470 <210> 37 <211> 515 <212> PRT <213> Artificial Sequence <400> 37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Val Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 355 360 365 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 370 375 380 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 385 390 395 400 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 405 410 415 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 420 425 430 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 435 440 445 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 450 455 460 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 465 470 475 480 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 485 490 495 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 500 505 510 Pro Pro Arg 515 <210> 38 <211> 473 <212> PRT <213> Artificial Sequence <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln 405 410 415 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 39 <211> 470 <212> PRT <213> Artificial Sequence <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460 Gln Ala Leu Pro Pro Arg 465 470 <210> 40 <211> 515 <212> PRT <213> Artificial Sequence <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 355 360 365 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 370 375 380 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 385 390 395 400 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 405 410 415 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 420 425 430 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 435 440 445 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 450 455 460 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 465 470 475 480 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 485 490 495 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 500 505 510 Pro Pro Arg 515 <210> 41 <211> 473 <212> PRT <213> Artificial Sequence <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln 405 410 415 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 42 <211> 470 <212> PRT <213> Artificial Sequence <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460 Gln Ala Leu Pro Pro Arg 465 470 <210> 43 <211> 515 <212> PRT <213> Artificial Sequence <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 355 360 365 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 370 375 380 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 385 390 395 400 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 405 410 415 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 420 425 430 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 435 440 445 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 450 455 460 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 465 470 475 480 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 485 490 495 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 500 505 510 Pro Pro Arg 515 <210> 44 <211> 471 <212> PRT <213> Artificial Sequence <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Lys Pro Val Leu Arg Thr 245 250 255 Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu 260 265 270 Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala 275 280 285 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu 290 295 300 Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg Ser Arg Lys 305 310 315 320 Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys Lys 325 330 335 Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys Pro 340 345 350 Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu Ser Arg Ser Ala 355 360 365 Glu Thr Ala Ala Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Glu Lys Lys Arg Ala 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Gln Gln Arg Arg Arg Asn Pro Gln 405 410 415 Glu Gly Val Tyr Asn Ala Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Thr Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Thr Leu Ala 465 470

Claims

1. The application of immune effector cells expressing receptors that recognize tumor antigens in the preparation of drugs, characterized in that, The drug contains the immune effector cells and gemcitabine for the treatment of tumors in human patients, wherein the immune effector cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the effects of the immune effector cells and gemcitabine used alone. The immune effector cells are chimeric antigen receptor (CAR) T cells; The CAR T cells recognize tumor cells expressing Claudin18.2; The tumor in question is pancreatic cancer.

2. The application as described in claim 1, characterized in that, The chimeric antigen receptor has: (i) Antibodies that recognize tumor antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD28, and CD3ζ; or (ii) Antibodies that recognize tumor antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD137, and CD3ζ; or (iii) Antibodies that recognize tumor antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD28, co-stimulatory signaling domains of CD137, and CD3ζ.

3. The application as described in claim 2, characterized in that, The amino acid sequence of the antibody that recognizes the tumor antigen is any one of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 14-22.

4. The application as described in claim 2, characterized in that, The amino acid sequence of the chimeric antigen receptor is any one of the amino acid sequences shown in SEQ ID NO: 23-44.

5. The application as described in claim 1, characterized in that, The T cells are selected from autologous T cells and allogeneic T cells.

6. The application as described in claim 5, characterized in that, The T cells mentioned are autologous T cells.

7. The application as described in claim 1, characterized in that, Gemcitabine is administered orally, intraperitoneally, or by injection.

8. A kit for treating tumors, characterized in that, The kit contains: 1) Immune effector cells that express receptors that recognize tumor antigens; 2) Gescitabine; 3) For containers containing 1) and 2) above; and 4) Instructions for use in treating tumors using the kit; The immune effector cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the effects of the immune effector cells and gemcitabine used alone; the immune effector cells are chimeric antigen receptor (CAR) T cells that recognize tumor cells expressing Claudin18.2, wherein the tumor is pancreatic cancer.

9. The reagent kit as described in claim 8, characterized in that, The chimeric antigen receptor has: (i) Antibodies that recognize tumor antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD28, and CD3ζ; or (ii) Antibodies that recognize tumor antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD137, and CD3ζ; or (iii) Antibodies that recognize tumor antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD28, co-stimulatory signaling domains of CD137, and CD3ζ.

10. The kit according to claim 9, characterized in that, The amino acid sequence of the antibody that recognizes the tumor antigen is any one of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 14-22.

11. The kit according to claim 9, characterized in that, The amino acid sequence of the chimeric antigen receptor is any one of the amino acid sequences shown in SEQ ID NO: 23-44.

12. The kit as described in claim 8, characterized in that, The T cells are selected from autologous T cells and allogeneic T cells.

13. The kit as described in claim 8, characterized in that, The T cells mentioned are autologous T cells.

14. The kit as described in claim 8, characterized in that, Gemcitabine is administered orally, intraperitoneally, or by injection.

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